The method of ion-exchange chromatography, in particular chromatography on modified silochromes (cf. USSR Inventor's Certificate No. 551339), is currently employed for the preparation of high-purity proteinases. This method however, is insufficiently selective, i.e. it does not enable the recovery of enzymes, in a high yield, directly from mixtures with a low concentration of the active enzyme containing a high amount of inorganic and organic impurities (e.g. from culture liquid).
For this reason, for the selective purification of enzymes the method of affine chromatography is more frequently employed. The method is based on the affinity of enzymes towards specific analogues of a substrate or inhibitors covalently bonded with the insoluble carrier thus making it possible to separate enzymes on the basis of their biological specificity and, consequently, obtain a higher degree of purification in comparison with other types of chromatographic techniques.
Known in the art are methods for purification of proteolytic enzymes comprising biospecific sorption on sorbents which are insoluble carriers, such as derivative of argarose, activated by means of a condensation agent, such as bromocyanogen, and covalently bonded with ligands. These substances are specific for a given class of enzymes such as gramicidin S, bacitracin, bacilliquine, phenylboric acid.
Sorption of enzymes on these sorbents is effected at a pH within the range of from 1.8 to 8.0 in salt buffers. Upon elution, an organic solvent is added to the salt buffer to ensure a complete desorption. The yield of enzymes in terms of activity is as high as 86%, the degree of purification is varied within the range of 2 to 95 times as compared to the starting material depending on its purity. However, sorbents produced, e.g. from sepharose which is a derivative of agarose contain 0.6 to 10 mcmol of the ligand per ml of a wet sorbent. This concentration of the ligand does not make it possible to ensure a maximum sorption of the enzyme per unit volume of the column, thus lowering the yield of the active enzyme per unit volume, which, in turn, lowers the process efficiency.
Furthermore, the above-mentioned sorbents have insufficient hydrodynamic properties. Thus, a solution of an enzyme is eluted from a column with 25 ml of phenylcarbonate sepharose at the rate of 10 ml/hr.
From a column with 28 of bacitracin-sepharose containing 10 .mu.mol of the ligand per ml of a wet sorbent, there are obtained, directly from 500 ml of a culture liquid of Actinomyces Sp., 10 mg per hour of enzyme purified by 104 times as compared to the starting material.
The sorbents based on agarose cannot be employed in purification of solutions of proteinases containing enzymes destroying agarose as impurities.
The use of a highly-toxic bromocyanogen for activation of agarose or derivatives thereof hinders the preparation of large amounts of sorbents, thus increasing production costs thereof.
It is an object of the present invention to provide a process for purification of proteolytic enzymes which features a higher productivity due to increased output with high yield of enzymes of high purity and activity per unit volume and per unit time.